The role of an amphiphilic helix and transmembrane region in the efficient acylation of the M2 protein from influenza virus

Protein palmitoylation, a cellular process occurring at the membrane-cytosol interface, is orchestrated by members of the DHHC enzyme family and plays a pivotal role in regulating various cellular functions. The M2 protein of the influenza virus, which is acylated at a membrane-near amphiphilic helix serves as a model for studying the intricate signals governing acylation and its interaction with the cognate enzyme, DHHC20. We investigate it here using both experimental and computational assays. We report that altering the biophysical properties of the amphiphilic helix, particularly by shortening or disrupting it, results in a substantial reduction in M2 palmitoylation, but does not entirely abolish the process. Intriguingly, DHHC20 exhibits an augmented affinity for some M2 mutants compared to the wildtype M2. Molecular dynamics simulations unveil interactions between amino acids of the helix and the catalytically significant DHHC and TTXE motifs of DHHC20. Our findings suggest that the binding of M2 to DHHC20, while not highly specific, is mediated by requisite contacts, possibly instigating the transfer of fatty acids. A comprehensive comprehension of protein palmitoylation mechanisms is imperative for the development of DHHC-specific inhibitors, holding promise for the treatment of diverse human diseases.

deprotonated.The resulting thiolate then acts as a nucleophile and attacks the carbonyl carbon of Pal-CoA 10,11 .To initiate the palmitoylation reaction Asp153 needs to polarise the neighbouring His154, which then acts as a base and deprotonates Cys156.The other His155 of the DHHC motive coordinates one of the two Zinc ions, which are important for the structural integrity of DHHC proteins 12 .Downstream of TM4 but spatially close to the DHHC-motif is a conserved 240 Thr-Thr-X-Glu 243 (TTXE) motif, which is also important for catalysis 4 .The structure of autoacylated DHHC20 shows that the side chain of Thr241 interacts with the carboxylate of Asp153, hindering its function for the deprotonation of His154 (Fig. 1).To transfer the fatty acid from DHHC to a substrate protein these hydrophilic interactions need to be remodelled.
The molecular mechanism by which substrates are recognized and enzyme catalysis is initiated is poorly understood.Several DHHCs contain interaction domains, such as ankyrin repeats, PDZ-binding motifs and SH2 domains with which they recruit (mainly intrinsically hydrophilic) substrates.Only one structure of such a complex is known, namely for an N-terminal ankyrin domain of DHHC17 bound to a peptide from the substrate SNAP 25 13 .How transmembrane proteins are recognized by DHHC enzymes is essentially unknown and there is no consensus sequence that directs palmitoylation 10,14,15 .However, there is increasing evidence that in various proteins an amphiphilic helix, often located near a transmembrane region, serves as a recognition feature for DHHC20 and other DHHCs, but no molecular structural information is available for any of these helices [16][17][18][19][20] .
Protein palmitoylation was first described for viral spike proteins and they have been important in elucidating basic biochemical features of S-acylation [21][22][23] .Here we used the short ion channel M2 of Influenza A virus to determine molecular determinants of palmitoylation.We have recently shown that DHHC 2, 8 15 and 20 are required for acylation of M2 and a similar set of DHHC proteins is involved in acylation of the spike glycoprotein hemagglutinin (HA) of the same virus 24 .M2 consists of a short N-terminal domain exposed extracellularly, a transmembrane segment of 19 amino acids and a 54-residue cytoplasmic tail.The M2 protein is expressed abundantly at the cell surface but present in only a few copies in virus particles 25,26 .During virus entry the proton channel activity of M2 acidifies the interior of virus particles which is required for uncoating of the viral genome 27 .During virus budding a membrane-near amphiphilic helix inserts into the cytosolic part of the plasma membrane, which causes membrane curvature required for virus scission 28 .The amphiphilic helix is palmitoylated at one cysteine at its beginning, but removal of the palmitoylation site only subtly affects virus replication [29][30][31] .The formation of the helix requires binding to lipid membranes, but not fatty acid attachment 32,33 .M2 is of interest since an NMR structure of the transmembrane region and amphiphilic helix is available 33 .This allows not only for targeted mutations but also to develop a model of the DHHC20/M2 complex.Deciphering the mechanism of protein palmitoylation and identifying the contact structures involved is important to develop DHHC-specific inhibitors that interfere with replication of many viruses and target a variety of other human diseases 34,35 .

The amphiphilic helix of M2 contains intrinsic signals for palmitoylation
A M2 monomer (97 amino acids) is composed of a short, unglycosylated ectodomain, one transmembrane region (TM, aa 26-43) and a cytoplasmic tail that contains an amphiphilic helix (AH, 47-61) and an intrinsically disordered region (62-80).M2 is a homo-tetramer, which is stabilized by formation of intersubunit disulphide bonds between Cys17 and Cys19 in the ectodomain (Fig. 2A). Figure 2B shows the NMR structure of the TM and AH integrated into a virtual lipid bilayer 33 .The four helical transmembrane domains run through the membrane at an angle and are slightly bent in the middle due to the presence of Gly34, and connected by a short 90° turn to the amphiphilic helix.The helix runs almost parallel to the membrane and contains the acylated Cys50 at the beginning of the hydrophobic face, so the fatty acid can insert into the lipid bilayer.The C-terminal parts of two TMs are connected by an ionic bond between Asp44 and Arg45 and two AHs by a hydrogen bond between Lys49 and Gly62 (Fig. 2C).The AH contains many amino acids with distinctive side chains, such as Phe, Tyr, His, Arg, Lys, which are localized on its surface and could bind to a DHHC enzyme (Fig. 2C and D).Note, however that the sequence of the AH is not conserved through all Influenza strains.The M2 protein we used contains two non-conservative (Phe54Arg, Glu56Arg) and one conservative amino acid exchange (His57Tyr, stained in cyan Fig. 1B) relative to the M2 for which the NMR structure was determined (Fig. 2E).It thus corresponds better to the consensus sequence calculated for all M2 proteins (Fig. 2F).
To investigate whether the helix of M2 becomes acylated in the absence of the TM, we fused its sequence, either corresponding to the wt helix or a Cys50Ser mutant, to the C-terminus of the red fluorescent protein (RFP).Confocal microscopy of transfected BHK21 cells revealed, that RFP, which has no intrinsic membrane targeting features, localizes to the cytosol and to the nucleus.In contrast, RFP-AH and RFP-AH-C50S are excluded from the nucleus and redistributed to intracellular compartments, especially to a perinuclear region, which is partially stained by antibodies against GM130, a component of the cis-Golgi, an intracellular site where DHHC20 (in addition to the ER) is located 36 (Fig. 3A).
We then performed a membrane fractionation assay to analyse the distribution of the proteins between cytosol and membranes.As expected, RFP is present only in the cytosolic fraction, whereas RFP-AH and also RFP-AH-C50S are also membrane-bound, ̴ 30% of total RFP-AH is in the membrane fraction (Fig. 3B).Thus, the amphiphilic helix of M2 has the capacity to interact with cellular membranes, even in the absence of the acylation site.However, quantification of three independent experiments revealed that membrane binding of RFP-AH-C50S is 35% reduced relative to RFP-AH (Fig. 3C).
To test whether RFP-AH becomes acylated, we used the Acyl-RAC (resin-assisted capture) assay, which exploits thiol-reactive resins to capture SH-groups in proteins.Twenty-four hours after transfection, cells were lysed and 10% of the total extract (TE) was removed from the lysate to determine the expression levels.Disulphide bonds in proteins present in the remaining part were reduced and newly exposed -SH groups were blocked.The sample was then equally split: one aliquot was treated with hydroxylamine (+ HA) to cleave thioester bonds, and the other aliquot was treated as control with Tris-HCl buffer (-HA).After pull-down of proteins with the thiol-reactive resin, samples were subjected to western blotting using antibodies against the RFP-tag.To exclude that proteins were lost during sample preparation, we used antibodies against the cellular palmitoylated protein flotillin 2 as an internal control.The results clearly show that RFP-AH is acylated, since it is precipitated by the thiol-reactive beads, which is not the case for RFP and RFP-AH-C50S (Fig. 3D).

Mutations in the helix reduce but do not abolish palmitoylation of M2
We then investigated which features of the helix are essential for acylation of M2.We used the authentic M2 protein, which is targeted by its transmembrane region to the ER and then transported through the exocytic pathway to the plasma membrane such that all molecules have access to DHHC enzymes 25,37 .We exchanged amino acids exposed at the molecule's surface, that alter the charge of the helix and/or its biophysical properties, such as hydrophobicity and hydrophobic moment.In M2 AH-1 the positively charged Lys49, Arg53 and Arg56 exposed at the frontside of the AH were exchanged by negatively charged Glu (Fig. 4A, see Supplementary Fig. 1 for localization of mutated amino acids in a surface representation of M2).These substitutions do not greatly alter the hydrophobicity and the hydrophobic moment of the helix as calculated by the tool heliquest, but they eliminate its positive net charge (Fig. 4B).In M2-AH-2, two long hydrophobic residues (Tyr52, Leu59) located at the back bottom of the AH were replaced by less hydrophobic alanine residues.In M2-AH-3, Phe47, which forms a distinct protrusion at the beginning of the helix near the acylated cysteine, has been replaced by alanine.The helices of M2-AH-2 and M2-AH-3 are less hydrophobic, but still exhibit an amphiphilic character.M2-AH-4 contains three positively charged lysine instead of three hydrophobic residues (Phe48, Ile51, Phe55), located at the back bottom of the helix.In AH-5, four positively charged residues (Lys49, Arg53, Lys60, Arg61), which are exposed at the frontside of the AH, were replaced by Phe.The mutations in M2-AH-4 and M2-AH-5 essentially destroyed the amphiphilic character of the helix.AH-4 is hydrophilic and AH-5 is more hydrophobic compared to the wt-helix.
The resulting mutants were expressed in 293 T cells, which were analysed by western blotting with M2 antibodies.The signals of M2-AH-1, M2-AH-3 and M2-AH-5 are substantially reduced compared to those of M2 wt and the other mutants (Supplementary Fig. 2).We were concerned that the mutations would lead to misfolding and subsequent degradation of M2 by the cell's quality control system, which would manifest as blockage of transport out of the ER 38 .Confocal microscopy revealed for M2 wt and all mutants reticular staining in the cytosol, likely representing the ER, transport to the plasma membrane and especially bright perinuclear staining, which overlaps with the cis-Golgi marker GM 130 (Fig. 4C).Thus, the M2 mutant proteins expressed are localized to the same compartments as M2 wt.Calculation of the Pearson's correlation coefficient revealed that four M2 mutants co-localize to the same extent as M2 wt with the marker GM130.Only transport of M2-AH-4 to the www.nature.com/scientificreports/cis-Golgi was slightly reduced (Supplementary Fig. 9).Therefore, the mutations are unlikely to cause misfolding of M2 and a defect in its transport to the intracellular acylation site.However, to compare the acylation of the M2-AH mutants, we had to adjust the amount of cell lysate used as input and accordingly the amount incubated with the thiol-reactive beads to account for the different expression levels.This prevented us from using flotillin as internal acylation control.Instead, we performed each Acyl-Rac assay four times to compensate for random loss of protein during sample preparation.Two of the results are shown in Fig. 4D to demonstrate the variability between experiments.The quantification of the five experiments (ratio of hydroxylamine-treated to input M2 normalized to M2 wt) is shown in Fig. 4E.Each mutation reduced acylation of M2 in each experiment.The reduction was greatest in M2-AH5 (reduced to 30% relative to M2 wt), followed by M2-AH-2 (50%), M2-AH-4 (54%) M2-AH-1 (62%) and least in AH-3 (75%).Although the differences between the experiments are relatively large, the mean values for most mutants (with the exception of M2 AH-3) were statistically significantly different from those for M2 wt.

Truncation and destruction of the helix reduces, but does not abolish palmitoylation of M2
We investigated next whether a helical structure downstream of Cys50 is required for the basal acylation of M2.We created two mutants where the cytoplasmic tail of M2 including most parts of the helix were deleted.Mutant M2 1-50 retains the first four helical residues including Cys50, M2 1-53 contains three additional residues downstream of the acylation site (Fig. 5A).M2 1-53 and especially M2 1-50 are expressed at lower levels than M2 wt (Supplementary Fig. 2).However, confocal microscopy revealed for both mutants the staining pattern typical for M2, including strong perinuclear staining.M2 1-50 even showed slightly enhanced co-localization with the cis-Golgi marker compared to M2 wt and M2 1-53 (Fig. 5B, quantification in Supplementary Fig. 9).The Acyl-RAC assay showed that the acylation of the mutants is greatly reduced but still clearly detectable (Fig. 5C).Quantification of four experiments showed a statistically significant reduction of about 45% for M2 1-50 and 40% for M2 1-53, but with relatively large variation between experiments (Fig. 5D).
Two further mutants were produced where we inserted helix-destroying proline residues.In the M2-1P mutant, Ile 51 following the acylated cysteine was replaced by a proline.The M2-3P mutant contains three prolines instead of Tyr52, Arg53 and Arg54 (Fig. 6A).Only mutant M2-1P was expressed at lower level (Supplementary Fig. 2), but it co-localizes with the cis-Golgi marker to the same extent as M2-3P and M2-wt (Fig. 6B, quantification in Supplementary Fig. 9).The Acyl-RAC assay showed that the acylation of the mutant M2-1P is greatly reduced to 30% relative to M2 wt.An effect of the insertion of three Pro is less obvious.The mean value of the degree of acylation from four experiments is 82%, but the results are not statistically significant different from M2 wt (Fig. 6C,D).
These results confirm that an amphiphilic helix is essential for efficient acylation of M2, but that a basal level of acylation is maintained even in the absence of a helical structure downstream of the acylation site.This suggests that amino acids in the transmembrane region of M2 might also affect the acylation reaction.

Replacement of kink-inducing glycine in the transmembrane helix increases palmitoylation of M2
The transmembrane region of M2 (sequence LVIAANIIGILHLILWIL) contains many of the hydrophobic amino acids commonly found in such a region, which are unlikely to be a specific recognition feature for a DHHC enzyme.Two particular and conserved residues are His37 and Trp41 but their side chains are pointing into the pore formed by tetrameric transmembrane region, and they are known to be essential for proton transport.One peculiar feature of the transmembrane region is a highly conserved glycine in its middle that causes a slight kink in the helix 33 .We changed the helix-disfavouring glycine to an alanine, a common amino acid in helical transmembrane regions.The resulting mutant M2-G34A is expressed at a higher level as M2 wt (Supplementary Fig. 2) and shows the same intracellular staining pattern in confocal microscopy (Fig. 6B, quantification in Supplementary Fig. 9).Quantification of four Acyl-Rac assays indicates that the acylation level of M2-G34A might be slightly higher than that of M2-wt (118%), but the difference was not statistically significant (Fig. 6C,D).We suspected that the relevance of the transmembrane region on acylation would become more pronounced if we minimise the influence of the helix.Thus, we introduced the G34A mutation into the truncated mutant M2 1-50 (Fig. 7A).Both proteins show a similar intracellular staining pattern in confocal micrographs (Fig. 7B).The Acyl-RAC assay shows that both proteins are expressed at the same level (input in Fig. 7c), but the M2-1-50-G34A band in the + HA samples is more intense than the M2-1-50 band.Quantification of three different experiments revealed a 50% increase in acylation of M2-1-50-G34A (Fig. 7D).

Mutations in the helix of M2 do not prevent binding to DHHC20
M2 wt substantially overlaps with DHHC20 in co-transfected BHK21 cells revealing mainly a reticular ERlike and perinuclear staining pattern (Supplementary Fig. 3).Some substrates can be co-immunoprecipitated with their cognate DHHC indicating a long-term, stable interaction during the enzymatic reaction 39 .We asked whether this also applies for the M2-DHHC20 complex and how the most severe helix mutations, M2-1P, M2-3P, M2-AH4 and M2-AH5 affect this interaction.Since the specificity of most commercially available antibodies against DHHCs have not been validated, we used a plasmid encoding human DHHC20 fused at its C-terminus to a myc-tag.DHHC20-myc and M2 were co-expressed in 293 T cells, which were lysed 24 h later.An aliquot of the lysate (5-10%) was removed to monitor the expression level (input).The remainder was subjected to immunoprecipitation with M2 antibodies and the precipitates were blotted with anti-myc and anti-M2 antibodies (Co-IP).Since the expression levels of some M2 mutants, especially M2-AH5, were lower, we adjusted the amount of lysate incubated with the anti-M2 antibodies to provide the same amount of protein.Roughly 1% of total DHHC20-myc was co-precipitated with M2-wt and the M2 mutants also interact with DHHC20-myc (Fig. 8A).The experiments were performed three times and the ratio of co-precipitated DHHC20-myc and M2 were calculated and normalized to the values of M2-wt (Fig. 8B).The results show that the introduction of helix-breaking proline residues does not affect the interaction with DHHC20-myc, but AH-4 and AH-5 have a clear but opposite effect on DHHC20 binding.It is reduced to ~ 20% for M2-AH-5, but increased around twofold for M2-AH-4.
Finally, we analysed the truncated M2 mutant 1-50 for binding to DHHC20-myc.Blotting the M2 antibody precipitate with myc antibodies shows the DHHC20-myc band, even stronger than the DHHC20-myc band obtained after co-expression with M2 wt.Surprisingly, no M2 1-50 was detected, (after longer exposure very little was found), when probing the same membrane with M2 antibodies, although both M2 wt and M2 1-50 are detected at similar levels in the input (Fig. 8C).However, when the M2 antibody precipitate was subjected to non-reducing SDS-PAGE, M2 1-50 can be clearly detected, mainly as a disulphide-linked dimer.M2 wt also appears mainly as a dimer, but also forms some disulphide-linked trimers and tetramers, as already described 40 .The DHHC20-myc band is also present in samples separated by non-reducing SDS-PAGE, and to a slightly higher extent if co-expressed with M2 1-50 (Fig. 8D).It thus appears that M2 1-50 forms unusual large aggregates during immunoprecipitation that fail to penetrate the gel under reducing conditions.Nevertheless, M2 1-50, which basically consists of one transmembrane region binds to DHHC20 inside cells.The remainder of the lysate was divided into two aliquots that were adjusted to the same extent.Samples were then subjected to immunoprecipitation with M2 antibodies and to reducing SDS-PAGE and blotting with anti-myc and anti-M2 antibodies.The Co-IP-blot probed with anti-myc antibodies was exposed 5 times longer than the input blot.(B) Quantification of this and two other independent experiments.One-way ANOVA was applied for statistical analysis.ns not significant, *P < 0.05, **P < 0.01 versus wild type.(C) DHHC20-myc and M2 wild type and M2 1-50 were expressed in 293 T cells.Cells were lysed with non-denaturating detergent, different volumes of the lysate were removed to adjust for the reduced expression level of M2 1-50 and samples were subjected to reducing SDS-PAGE and blotting with anti-myc and anti M2 antibodies.Co-IP: The remainder of the lysate was divided into two aliquots that were adjusted to the same extent.Samples were then subjected to immunoprecipitation with M2 antibodies and to reducing SDS-PAGE and blotting with anti-myc and anti-M2 antibodies.(D) Co-IP from C subjected to non-reducing SDS-PAGE.
The experimental results are summarized in Supplementary Table 1.We conclude that most M2 mutants can form an enzyme-substrate complex, but that the subsequent steps of the enzymatic reaction, fatty acid transfer or, in the case of mutant AH4, perhaps release of the M2 protein from the enzyme, are impaired.

Molecular modelling of the M2 DHHC 20 interaction
To identify the contact surface between M2 and DHHC20 we performed molecular dynamics simulations.A fully resolved docked structure is currently unknown so we follow the steps outlined in the methods section to generate an initial starting representation.A snapshot of the simulation of the M2 DHHC20 complex is shown as a surface representation in Fig. 9A.The figure shows that the amphiphilic helix of M2 docks to a 25 Å wide cavity in the cytoplasmic domain of DHHC20 close to its transmembrane region.Contact analysis, as described in the methods section, shows that the proteins form 18 interactions involving 13 residues in DHHC20 and 12 residues in M2 (highlighted in both structures in Supplementary Fig. 4 and listed in Supplementary Table 2).These interactions can be broken into interactions residing in the transmembrane and amphiphilic sections of M2.In the transmembrane helix four residues of M2 interact with four residues located on the outside of TM1 of DHHC20.Ile39 of M2 also interacts with Trp158, which is located near the hydrophobic tunnel and is supposed to act as a gate (Fig. 9B) 41 .
We also performed MD simulations with different M2 mutants to observe structural differences in binding sites, either by deletion of the amphiphilic helix or single amino acid substitutions as described in the methods section.As we have no direct way with classical MD simulations to observe the transfer of the fatty acid from DHHC to M2, the distance between M2 Cys50 and DHHC Cys156 is used as a proxy for binding affinity.If the www.nature.com/scientificreports/distance between the residues remains within the bounds of thermal fluctuations in comparison to the wt, we can suspect that transfer could occur on the given timescale.A plot of this Cys-Cys distance as a function of simulation length is shown in Supplementary Fig. 5A and we observe that for the M2 1-50 (red line) this distance jumps to values to over 15 Å over the course of 100 ns and drifts to 20 Å in the second half of the simulation.Examination of the trajectory shows that the truncated M2 molecule starts to align along the transmembrane domain of DHHC20 (Supplementary Fig. 5B) forming new contacts listed in Supplementary Table 3.The M2 Ile51Pro (orange) and Ile51Ala (green) mutants remain within a similar range (7-10 Å) to the wt.While the distance is conserved observation of the trajectories highlights a decrease in angle between the transmembrane region and the helix which we attribute to the changed geometry of the M2 mutants (Supplementary Fig. 6).Contact analysis shows that the contact of Phe42 in M2 with His 154/155 of the DHHC motif is shifted to preferential contacts with Pro157/Trp158 in Ile51Pro and in Ile51Ala.Likewise, the contact between Arg53 of M2 and Thr241 of the TTXE motive is reduced by ~ 60/30% in the Ile51Pro/Ile51Ala relative to the wt (Supplementary Table 4).
In addition to the Cys-Cys distance analysis, we directly estimate the free energy of binding for a Arg53Ala mutant by construction of a thermodynamic cycle as outlined in the method section.This residue was selected for mutation as Arg53 shows a high degree of contact from the wt trajectory and is in part responsible for the amphiphilic nature of the helix.In addition, the AH-5 experimental mutation showed substantial reduction in expression.However, we observe that strikingly the Arg53Ala mutant has an increased binding affinity to DHHC over wt by 3.0 ± 0.7 kcal/mol.A similar contact analysis at the edges of the switching parameter show that the Arg53Ala mutation has generally stronger contacts overall, in particular for residues Phe47, Cys50, and Arg54 (Supplementary Table 5).Analysis of the radius of gyration of the M2 only trajectory, i.e. no DHHC present, show that the wt prefers to adopt a more extended structure ( R g = 17.89 ± 0.09 Å) but the Arg53Ala mutant more readily adopts the compact structure ( R g = 17.3 ± 0.3 Å) that is more indicative of the M2/DHHC complex (Supplementary Fig. 7).

The amphiphilic helix of M2 contains all the required information for palmitoylation
The amphiphilic helix of M2 becomes palmitoylated when fused to the C-terminus of the red fluorescent protein indicating that the information for fatty acid attachment is encoded in the helix.The helix also redistributes the reporter protein from its usual nuclear and cytosolic location to cellular membranes.This also occurs, although is reduced by about 30%, when the acylation site is replaced by a serine (Fig. 3).Thus, the helix of M2 has intrinsic propensity to associate with cellular membranes, which is then stabilized by palmitoylation 28,42 .A substantial proportion of RFP-AH (~ 70%) is located in the cytosol, making it unlikely that acylation is stochiometric, since palmitoylated molecules are (almost) always membrane-bound.There are several mutually exclusive explanations for this observation: some of the fatty acids are cleaved by thioesterases, the initial palmitoylation is substoichiometric because the AH does not contain all the information for complete acylation, and/or due to incomplete targeting to membranes not every molecule has access to the membrane-bound DHHC.

Mutations in the amphiphilic helix reduce, but do not eliminate palmitoylation of M2
To further investigate the signals in the AH, we used the full-length M2 protein, as it is transported to the plasma membrane via the exocytic pathway and thus has complete access to DHHC enzymes 25 .As described previously 43 some of the mutations reduced the expression level of M2 but this is unlikely to be due to misfolding, since all mutants were transported along the exocytic pathway and co-localized with a cis-Golgi marker to the same extent as M2 wt.Each mutation we introduced into the amphiphilic helix reduced the acylation of M2, but to varying degrees, from 30 to 82% compared to M2 wt (Figs. 4, 5, 6, results are summarized in Supplementary Table 1).The strongest reduction to 30% was achieved by eliminating the hydrophobic moment of the AH, increasing its hydrophobicity (M2 AH-5), and by inserting a proline adjacent to the acylated cysteine (M2-1P).The latter mutant still contains all amino acids identified by molecular dynamics to interact with DHHC20.In M2 AH-4, none of the interacting amino acids were exchanged either, although acylation was determined to be reduced by about 50%.This indicates that the conformation of the helix and its biophysical properties, such as hydrophobicity and hydrophobic moment play a role for efficient acylation.Note, that the inherent variability of the Acyl-RAC assay makes it difficult to assess whether certain mutations have stronger effects than others.The acylation level of two mutants, M2 AH-3 (to 74%) and M2-3P (to 82%), was not statistically significantly different from M2 wt, although the reduction was seen in (almost) every experiment.It is therefore safe to conclude, that any of the mutations we introduced into the AH, reduces but does not eliminate fatty acid transfer to M2.Even after removal of all residues beyond Cys50, M2 is still about 50% acylated compared to M2 wt.This suggests that the amino acids preceding the acylation site also affect acylation.

The transmembrane region effects acylation of M2
Replacing a kink-inducing glycine in the middle of the transmembrane helix with an alanine increased palmitoylation, which was statistically significant in the context of the M2 1-50 mutant (Fig. 7).Glycine is not one of the residues identified by MD simulations to contact DHHC20, but it does cause a small kink in the middle of the helix 33 .We therefore suggest that the altered conformation of the transmembrane region presents the cysteine in a more favourable way to the active site of the acyltransferase.Note also that the acylated cysteine is always at position 50 in each variant of M2, suggesting that its position plays an important role.This is in contrast to the acylated cysteines in the cytoplasmic domain of the spike proteins of corona and influenza viruses, whose positions have shifted during viral evolution 44 .
The molecular dynamics simulation revealed that several residues of M2 wt are in the immediate vicinity to functionally important amino acids in DHHC20.Ile39 at the cytosolic end of the transmembrane region of M2, interacts with the Trp158 which is located near the opening of the hydrophobic cavity (Fig. 9B).The side chain of Trp158 was found in another molecular dynamics study to adopt two conformations, an open and a closed state, and it was therefore proposed that Trp158 acts as a gate for the hydrophobic cavity 41 .Thus, one can speculate that this interaction opens the gate for fatty acid transfer to M2.
Phe47 at the beginning of the amphiphilic helix is in close proximity to His154 whereas Cys50 contacts both Asp153 and His154 of the 153 DHHC 156 motif (Fig. 9B).One might speculate that His154 of DHHC20 deprotonates Cys50 to form the reactive thiolate which then can attack the thioester linkage between Cys156 and the fatty acid.In accordance, mutation of this histidine in the DHHC motif of the yeast enzyme Erf2 abolished palmitate transfer to the Ras2 substrate 9 .However, to act as a base His154 must be polarized by the neighbouring Asp153.The crystal structure of the autoacylated form of DHHC20 shows that Asp153 interacts with Thr241 of the TTXE motif hindering its function for the deprotonation of His154.In addition, His154 interacts with the carboxylate of the palmitoylation inhibitor 2-bromopalmitate, which is covalently bound to Cys156 via C2 (Supplementary Fig. 8).
MD simulations showed that Arg53 of M2 contacts Thr241 of the TTXE motif and the two adjacent amino acids Ser244 and Phe245 (Fig. 9C).It is thus tempting to speculate that this interaction could compete with the intramolecular interaction of Thr241 with Asp153 observed in the autoacylated form of DHHC20.This would release the carboxyl group of Asp153 to deprotonate His 154 and thus initiate the fatty acid transfer from DHHC20 to M2.
MD simulations of mutant Arg53Ala corroborate this speculation by examining the distance between the sidechain oxygen of Thr241 and the C γ of Asp153 in both the wt and mutant as shown in Supplementary Fig. 7.While the contact of residue 53 to Thr241 of the TTXE motif is maintained, Ala (unlike Arg) cannot form a hydrophilic interaction with Thr241 and thus the Arg53Ala mutant has a distance distribution shifted lower that favours an interaction with Asp153.This then prevents the proposed shifting of hydrogen bonds between the TTXE and DHHC motif.Likewise, exchange of Ile51 by Pro (and to a lesser extent by Ala) decreases the angle between the TM and the amphiphilic helix of M2.This altered geometry causes Phe47 to lose contact with His154, which needs to be deprotonated to act as a base.These altered interactions might explain the reduced acylation of mutant M2 1P although binding to DHHC20 is not affected.
In summary, we have shown that M2 requires an amphiphilic helix for efficient acylation.Based on MD simulations we speculate that specific interactions of amino acids in the helix of M2 with the DHHC and TTXE motif of DHHC20 initiate the fatty acid transfer.However, since several of the mutants contain exchanges in residues that do not contact DHHC20, general biophysical features of the helix, such as charge, hydrophobicity and hydrophobic moment play a crucial role for efficient acylation.Evidence of this is seen as M2 with mutations in the amphiphilic helix can still bind to DHHC20 but the subsequent step, the transfer of the fatty acid to M2, is impaired.
Cellular proteins are also acylated by DHHC20 on a cysteine at one end of an amphiphilic helix, usually localized near a membrane-spanning region [16][17][18]45 . Thee is no sequence similarity between these helices, suggesting that DHHC20 can acylate a variety of helical structures if they fit geometrically into the depression underneath the membrane-spanning region and have the correct biophysical properties.DHHC2, 8 and 15, which are also involved in acylation of M2 24 contain the same cavity, and the superposition of their structures shows that the DHHC and TTXE motifs are perfectly aligned (data not shown) suggesting that the same considerations also apply for these DHHCs.DHHC20 efficiently acylates typical viral spike proteins that do not have an amphiphilic helix in their cytoplasmic tail 24,46 and hence the enzyme might recognize other features in substrate proteins.It is not uncommon that the same DHHC exhibits more than one mode of enzyme-substrate interactions 47 .This is mediated by interactions between transmembrane regions, since the M2 1-50 mutant, which lacks most of the amino acids proposed to initiate fatty acid transfer, is still acylated.However, it is also conceivable that the basal acylation is due to a non-enzymatic fatty acid transfer.This has been described for purified proteins in the presence of Pal-CoA, but only at basic pH to deprotonate the cysteine.However, it also occurs in membranes at physiological pH, www.nature.com/scientificreports/albeit less efficiently than the corresponding enzymatic reaction 48 .The stable interaction with DHHC20 could lower the pKa of Cys50 in M2, which then spontaneously attacks the carbonyl carbon of Pal-CoA 49 .Purification of substrate and DHHC enzymes and in vitro reconstitution of the acylating activity are required to clarify these open questions 50 .

Cell lines, genes and plasmids
HEK293T cells and BHK21 cells, initially obtained from the American Type Culture Collection (ATCC number CRL-11268 and CCL-10, respectively) were grown in Dulbecco's modified Eagle's medium (DMEM, PAN Biotech) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C with 5% CO2.293 T cells were used to express the protein and test the S-acylation level.BHK21 cells were used for the immunofluorescence experiment.The M2 and M2 1-50 from human Influenza A virus A/WSN/33 (H1N1) (GenBank: CY034133.1)and RFP was cloned from LV-RFP (addgene: #26001with XhoI and BgIII (NEB) into the PCAGGS expression plasmids.M2 mutants were generated with quick change site-directed mutagenesis method with S7 Fusion Polymerase from Mobidiag.The clones of the RFP-AH and RFP-AH-C50S were made using restriction cloning with XhoI and BgIII (NEB) into the PCAGGS, the reverse primer encode the sequence of AH and AH-C50S of M2.Sequences of the primers are listed in the supplementary materials.The human DHHC20 gene equipped with a C-terminal myc-tag and cloned into pcDNA3.1 was provided by the Fukata lab 51 .

Membrane separation experiment
3 µg plasmids encoding RFP, RFP-AH or RFP-AH-C50S were transfected into 293 T cells using lipo3000 transfection reagent.After 24 h, the subcellular protein fractionation kit for cultured cells was used to separate cells into cytoplasm and membranes.All the buffers were prepared with protein inhibitors from the kit in advance.Cells (1 × 10 6 ), PBS-washed and pelleted in a microfuge (500×g, 5 min), were incubated with 150ul of the cytoplasmic extraction buffer CEB at 4 °C for 10 min with gentle mixing.This buffer permeabilizes the plasma membrane and releases the cytosol.The opened cells are pelleted (500×g, 5 min) and the supernatant (cytosol) is removed.150 ul membrane extraction buffer MEB was added to the pellet, first vortex the tube for 5 s and incubate tube at 4 °C for 10 min with gentle mixing This dissolves all membranes with the exception of the nuclear membrane.Centrifugation (3000×g for 5 min) pellets the nuclei, the supernatant contains extracts from the plasma membrane, mitochondria, and ER/Golgi membranes.10% of the cytosol preparation and 20% of the membranes were analysed by western blotting using anti-RFP antibodies (1:1000) and secondary anti-rabbit IgG VHH single domain antibody (1:3000) as described below.

Co-Immunoprecipitation experiments
Plasmids (1.5 µg) encoding M2-wt or M2 mutants were co-transfected with the plasmid (1.5 µg) encoding human DHHC20 fused at its cytosolic C-terminus to a myc tag into 293 T cells grown in 6-well plates.24 h after transfection cells were lysed with 250 µl NP40 lysis buffer (final concentration 0.5%) diluted in IP buffer (500 mM Tris-HCl, 20 mM EDTA, 30 mM sodium pyrophosphate decahydrate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM benzamidine, 1 mM PMSF, 1 mM NEM) with protein inhibitor for 1 h on ice.Cell debris were removed by centrifugation for 10 min at 10,000 rpm in a table top centrifuge.To determine the expression level (input control) 10% of the resulting supernatant was removed and subjected to western-blotting with either anti-M2 monoclonal antibody from mice (1:4000) or anti-myc-tag polyclonal antibody from rabbit (1:2000).
1 µl of the M2 antibody (diluted in 200ul 3% BSA buffer) was added to the remaining supernatant and incubated for 1 h at 4 °C with shaking.Later 30 µl of protein-G Sepharose 4 Fast Flow (GE Healthcare, prepared according to manufacturer instructions) were added to the samples and incubated at 4 °C with shaking overnight.Samples were then centrifuged for 5 min at 5000 rpm and the supernatant was removed.After three times washing with IP buffer, the pellet was dissolved in 40 µl of 2X SDS-PAGE loading buffer and subjected to western-blotting, first with anti-myc-tag antibody and secondly with anti-M2 antibody using the appropriate secondary antibodies as described below.The relative amount of DHHC20-myc coprecipitated with the M2

Figure 1 .
Figure 1.Catalytic centre of the autoacylated form of DHHC20.The amino acids of the DHHC motif and T241 of the TTXE motif are shown as sticks.Incubation with 2-Br-palmitate resulted in alkylation of cysteine 156 through attachment at the α position of palmitic acid.Zn zinc ion.Created with PyMol from pdb-file 6BML.

Figure 2 .
Figure 2. Primary and 3D structure of M2 of Influenza A virus.(A) Scheme of M2: Unglycosylated ectodomain, TM: transmembrane region (aa 26-43), AH: amphiphilic helix (47-61) cytoplasmic tail.(B) NMR structure of M2 embedded in a virtual lipid bilayer for visualization of the location of the TM and AH.G34: glycine in the middle of the TM slightly bends the helix.C50: acylation site at the beginning of the amphiphilic helix (AH).Note that the M2 used to determine the structure contains a serine instead of a cysteine at position 50.The figure was created from pdb file 2L0J with the PPM 3.0 web-server https:// opm.phar.umich.edu/ ppm_ serve r3, which calculates the position of a membrane protein within a lipid bilayer.(C) Location of amino acid side chains in the structure of the amphiphilic helix.The three residues highlighted in cyan differ between the M2 variants used for NMR and for our experiments.Hydrophilic interactions between D44 and R45 and K49 and G62 connect two monomers.(D) Surface representation of the amphiphilic helix.Charged amino acids in blue, aromatic in wheat and acylated Cys50 in red.(E) Amino acid sequence of the AH in M2 used for structural analysis (upper row) and in this study (lower row).(F) Web-Logo of all M2 sequences shows conserved and variable residues in the amphiphilic helix.

Figure 3 .Figure 4 .
Figure 3. Membrane localization and acylation of the amphiphilic helix of M2 fused to RFP. (A) Confocal microscopy: RFP, RFH-AH, RFP-AH-C50S were expressed in BHK21 cells, which were analysed by confocal microscopy.The scale bar is 20 µm.(B) Membrane separation experiment: RFP, RFH-AH, RFP-AH-C50S were expressed in 293 T cells.Cells were lysed and separated into soluble (S) and membranous (M) fractions, which were subjected to blotting with anti-RFP antibodies.10% of the cytosol preparation and 20% of the membranes were analysed in the blot.(C) Quantification of this and two other independent experiments.The ratio of the density of the M and S bands was calculated, normalized to RFP-AH (= 1).The mean ± SD and the results from the three independent experiments are shown.One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis.*P < 0.05 P = 0.0195, ****P < 0.0001 versus RFP-AH.(D) S-acylation:RFP, RFH-AH, RFP-AH-C50S were expressed in 293 T cells which were lysed 24 h after transfection.To test for protein expression, 10% of the lysate was removed (input).The remainder was divided into two aliquots, one not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group.Samples were subjected to Western blotting with antibodies against RFP and, subsequently against flotillin-2, an endogenous acylated protein.

Figure 5 .
Figure 5. Palmitoylation and intracellular localisation of M2 mutants with truncated amphiphilic helix.(A) Scheme and sequence of M2, wild type and truncated mutants.(B) Confocal microscopy of mutants in transfected BHK21 cells.Cells were permeabilized and stained with antibodies against M2 (green), the cis-Golgi marker GM130 (red) and with DAPI (blue) to highlight the nucleus.The scale bar is 20 µm.(C) S-acylation: M2 wt and the mutants were expressed in 293 T cells.For the input samples, different volume of the lysate was removed to adjust for the reduced expression level of some mutants.The remainder was divided into two aliquots that were adjusted to the same extent.One aliquot was not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group.Samples were subjected to Western blotting with antibodies against M2.The results of two independent experiments are shown.(D) Quantification of these two and two other independent experiments.The density of the bands with hydroxylamine (+ HA) bands was divided by the density of the input bands and normalized to wild type (= 1)).The mean ± SD and the results from four independent experiments are shown.One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis.**P < 0.01 versus wild type.

Figure 6 .
Figure 6.Palmitoylation and intracellular localization of M2 mutants with disrupted amphiphilic helix and replacement of a glycine in the transmembrane region.(A) Scheme and sequence of M2, wild type and mutants with one or three Pro inserted into the helix and with the Gly34Ala exchange in the TM.(B) Confocal microscopy of mutants in transfected BHK 21 cells.Cells were permeabilized and stained with antibodies against the cis-Golgi marker GM130 (red) and with DAPI (blue) to highlight the nucleus.The scale bar is 20 µm.(C) S-acylation: M2 wt and the mutants were expressed in 293 T cells.For the input samples, different volume of the lysate was removed to adjust for the reduced expression level of some mutants.The remainder was divided into two aliquots that were adjusted to the same extent.One aliquot was not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group.Samples were subjected to Western blotting with antibodies against M2.The results of two independent experiments are shown.(D) Quantification of 5C and two other independent experiments.The density of the bands with hydroxylamine (+ HA) bands was divided by density of the input bands and normalized to wild type (= 1)).The mean ± SD and the results from four independent experiments are shown.One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis.ns not significant, ***P < 0.001 versus wild type.

Figure 7 .
Figure 7. Palmitoylation and intracellular localization of M2 mutants with truncated amphiphilic helix and replacement of a glycine in the transmembrane region.(A) Scheme and sequence of M2 1-50, wild type and mutants with the Gly34Ala exchange in the TM.(B) Confocal microscopy of mutants in transfected BHK cells.Cells were permeabilized and stained with antibodies against the cis-Golgi marker GM130 (red) and with DAPI (blue) to highlight the nucleus.The scale bar is 20 µm.(C) S-acylation: M2 1-50 and M2 1-50-G34A were expressed in 293 T cells.For the input samples, same volume of the lysate was removed to detect expression level.The remainder was divided into two aliquots.One aliquot was not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group.Samples were subjected to Western blotting with antibodies against M2 and, subsequently against flotillin-2, an endogenous acylated protein.(D) Quantification of 6C and two other independent experiments.The density of the samples with hydroxylamine (+ HA) bands was divided by density of the input and normalized to wild type (= 1)).The mean ± SD and the results from three independent experiments are shown.Unpaired t-test was applied for statistical analysis.*P < 0.05, versus M2-1-50.

Figure 8 .
Figure 8. Co-precipitation of DHHC20 with M2 wt and M2 mutants with reduced acylation.(A) Input: DHHC20-myc and M2, either wild type and the indicated mutants were expressed in 293 T cells.Cells were lysed with non-denaturating detergent, different volumes of the lysate were removed to adjust for the reduced expression level of especially M2 AH-5 and samples were subjected to reducing SDS-PAGE and blotting with anti-myc and anti M2 antibodies.Co-IP: The remainder of the lysate was divided into two aliquots that were adjusted to the same extent.Samples were then subjected to immunoprecipitation with M2 antibodies and to reducing SDS-PAGE and blotting with anti-myc and anti-M2 antibodies.The Co-IP-blot probed with anti-myc antibodies was exposed 5 times longer than the input blot.(B) Quantification of this and two other independent experiments.One-way ANOVA was applied for statistical analysis.ns not significant, *P < 0.05, **P < 0.01 versus wild type.(C) DHHC20-myc and M2 wild type and M2 1-50 were expressed in 293 T cells.Cells were lysed with non-denaturating detergent, different volumes of the lysate were removed to adjust for the reduced expression level of M2 1-50 and samples were subjected to reducing SDS-PAGE and blotting with anti-myc and anti M2 antibodies.Co-IP: The remainder of the lysate was divided into two aliquots that were adjusted to the same extent.Samples were then subjected to immunoprecipitation with M2 antibodies and to reducing SDS-PAGE and blotting with anti-myc and anti-M2 antibodies.(D) Co-IP from C subjected to non-reducing SDS-PAGE.

Figure 9 .
Figure 9. Molecular dynamics simulation of the M2 DHHC20 complex.(A) Surface representation: M2 in red, AH: amphiphilic helix.DHHC20 in blue.CRD: cysteine-rich domain in the cytoplasmic domain.TM1: transmembrane region 1.See Supplementary Fig. 4 for a visualization of the interacting amino acids in the entire molecule of DHHC20 and M2.(B-D) Residues in the amphiphilic helix of M2 (blue) that contact residue in DHHC20 (green) important for catalysis.Residues in M2 are highlighted as cyan sticks and in DHHC as white sticks.The distance is indicated in angstroms.Orange stick: Fatty acid.Zn: Zinc ion.One snapshot of the MD simulation is shown.Note that the helix has a kink downstream of Cys50 which appears to remain during the course of the simulation. https://doi.org/10.1038/s41598-023-45945-z